Search

PURPOSE: Overlapping clinical features of cutaneous leishmaniasis (CL) with ulcers caused by fungi and mycobacteria necessitate confirmatory diagnostic testing. We evaluated a handheld battery-operated device for detection of CL and common fungal and mycobacterial causes of ulcers. METHODS: We validated Palm PCR™ for detection of common ulcerative skin pathogens using ATCC(®) reference and clinical strains of Leishmania, mycobacteria, and fungi in the lab and field. Amplified products were Sanger sequenced. Performance characteristics were calculated using conventional PCR as a reference standard. RESULTS: Palm PCR™ detected 100% of ATCC(®) strains of Leishmania, fungi, and mycobacteria, with sensitivity and specificity of 90% and 91.7%, respectively. In the field, the sensitivity for detection of Leishmania in patients with suspected CL was 100%. In 61% of CL patients, co-colonization with genera such as Malassezia, Aspergillus, Candida, and Cladosporium was detected. In 50% of CL patients with an inflammatory (secondarily infected) phenotype, detected fungal species had known associations with human cutaneous disease. CONCLUSIONS: Palm PCR™ performs comparably to conventional PCR for detection of Leishmania, fungi, and mycobacteria. This work has implications for the diagnostic approach to tropical ulcers, and has the potential to improve field detection of ulcerative pathogens in resource constrained areas

Background SARS-CoV-2 infection can present with a broad clinical differential that includes many other respiratory viruses; therefore, accurate tests are crucial to distinguish true COVID-19 cases from pathogens that do not require urgent public health interventions. Co-circulation of other respiratory viruses is largely unknown during the COVID-19 pandemic but would inform strategies to rapidly and accurately test patients with respiratory symptoms. Methods This study retrospectively examined 298,415 respiratory specimens collected from symptomatic patients for SARS-CoV-2 testing in the three months since COVID-19 was initially documented in the province of Alberta, Canada (March-May, 2020). By focusing on 52,285 specimens that were also tested with the Luminex Respiratory Pathogen Panel for 17 other pathogens, this study examines the prevalence of 18 potentially co-circulating pathogens and their relative rates in prior years versus since COVID-19 emerged, including four endemic coronaviruses. Results SARS-CoV-2 was identified in 2.2% of all specimens. Parallel broad multiplex testing detected additional pathogens in only 3.4% of these SARS-CoV-2-positive specimens: significantly less than in SARS-CoV-2-negative specimens (p p Conclusions Broad respiratory pathogen testing rarely detected additional pathogens in SARS-CoV-2-positive specimens. While helpful to understand co-circulation of respiratory viruses causing similar symptoms as COVID-19, ultimately these broad tests were resource-intensive and inflexible in a time when clinical laboratories face unprecedented demand for respiratory virus testing, with further increases expected during influenza season. A transition from broad, multiplex tests toward streamlined diagnostic algorithms targeting respiratory pathogens of public health concern could simultaneously reduce the overall burden on clinical laboratories while prioritizing testing of pathogens of public health importance. This is particularly valuable with ongoing strains on testing resources, exacerbated during influenza seasons.

Introduction: Leishmaniasis is a neglected tropical disease that manifests as three major disease phenotypes: cutaneous, mucocutaneous, and visceral. In this preliminary study, we quantified virulence factor (VF) RNA transcript expression in Leishmania species, stratified by geographic origin and propensity for specific disease phenotypes. Methods: Cultured promastigotes of 19 Leishmania clinical and ATCC isolates were extracted for total cellular RNA, cDNA was reverse transcribed, and qPCR assays were performed to quantify VF RNA transcript expression for hsp23, hsp70, hsp83, hsp100, mpi, cpb, and gp63. Results: Comparison of visceralizing species (Leishmania donovani, Leishmania chagasi, and Leishmania infantum) versus non-visceralizing species [Leishmania (Viannia) spp., Leishmania tropica, Leishmania major, Leishmania mexicana, and Leishmania amazonensis] revealed a significantly greater pooled transcript expression for visceralizing species (p = 0.0032). Similarly, Old World species demonstrated significantly higher VF RNA transcript expression than New World species (p = 0.0015). On a per-gene basis, species with a propensity to visceralize ubiquitously expressed higher levels of gp63 (p = 0.005), cpb (p = 0.0032), mpi (p = 0.0032), hsp23 (p = 0.0039), hsp70 (p = 0.0032), hsp83 (p = 0.0032), and hsp100 (p = 0.0032). Conclusion: Here, we provide quantitative, preliminary evidence of elevated VF RNA transcript expression driven largely by the visceralizing causative species of Leishmania. This work highlights the extensive heterogeneity in pathogenicity mechanisms between Leishmania species, which may partly underpin the fatal progression of visceral leishmaniasis.

Point-of-care dengue diagnostics are unavailable in most settings; thus, diagnosis is clinical until more definitive microbiological testing — such as serology — is resulted. Thrombocytopenia and lymphopenia are common hallmarks of dengue fever; however, neutropenia is a prominent, yet less frequently reported trend. We aimed to identify hematological patterns that can assist frontline clinicians with diagnostic certainty of dengue. Dengue patients presenting to our unit via the Emergency Department were compared to those presenting with other febrile illness (OFI) diagnoses. Patient demographics, day of illness, and neutrophil, lymphocyte, and platelet counts from days 1 to 14 of illness were collected, where available. Analyses were stratified by day of illness. Eighteen patients were included in the dengue group and 151 in the OFI group. The frequency of thrombocytopenia, neutropenia, and lymphopenia was each significantly greater in the dengue cohort than in the OFI group (p

The Zika virus outbreak in the Americas prompted enhanced global surveillance due to the recognition of adverse neurological outcomes, including Guillain-Barre Syndrome, a novel but devastating congenital syndrome. Symptomatic Zika virus infection is clinically indistinguishable from other arboviral infections of the Americas such as chikungunya, dengue, and the more recently emerging Mayaro virus. We conducted laboratory surveillance for such arboviruses in ill returned travelers to the Americas between November 2015 and May 2016 by examining whole blood, malaria-negative specimens with noted travel history to the Americas by real-time and end-point PCR for the following targets: chikungunya (CHIKV), flaviviruses (Pan-Flavi), dengue virus types 1–4 (DEN1, DEN2, DEN3, DEN4), and Zika virus (ZIKV). End-point PCR was performed to detect Mayaro virus (MAYV). 1294 malaria-negative specimens were evaluated for compatible travel history. Of 224 enrolled specimens, 124 (55%) were from returned travelers to the Caribbean, while 66 (29.5%) were from Central America, and 34 (15.2%) from South America. Arboviral pathogens detected included: dengue (n = 3, 1.3%), Zika virus (n = 2, 0.9%), and unspecified flavivirus (n = 2, 0.9%). No cases of chikungunya or Mayaro were detected in the cohort. Common arboviruses circulating in the Americas were detected in over 3% of whole-blood malaria-free specimens from ill returned travelers, supporting the notion that during this time period, Zika virus was as commonly imported as dengue from this geographic region. Surveillance for other flaviviruses and alphaviruses including Mayaro virus is warranted to detect sentinel events, and inform priorities for diagnostic testing capacity.

Leishmaniasis is a neglected tropical disease that manifests as three major disease phenotypes: cutaneous, mucocutaneous, and visceral. In this preliminary study, we quantified virulence factor (VF) RNA transcript expression inCultured promastigotes of 19Comparison of visceralizing species (Leishmania donovani, Leishmania chagasi, and Leishmania infantum) versus non-visceralizing species [Leishmania (Viannia) spp., Leishmania tropica, Leishmania major, Leishmania mexicana, and Leishmania amazonensis] revealed a significantly greater pooled transcript expression for visceralizing species (p = 0.0032). Similarly, Old World species demonstrated significantly higher VF RNA transcript expression than New World species (p = 0.0015). On a per-gene basis, species with a propensity to visceralize ubiquitously expressed higher levels of gp63 (p = 0.005), cpb (p = 0.0032), mpi (p = 0.0032), hsp23 (p = 0.0039), hsp70 (p = 0.0032), hsp83 (p = 0.0032), and hsp100 (p = 0.0032).Here, we provide quantitative, preliminary evidence of elevated VF RNA transcript expression driven largely by the visceralizing causative species of

Overlapping clinical features of cutaneous leishmaniasis (CL) with ulcers caused by fungi and mycobacteria necessitate confirmatory diagnostic testing. We evaluated a handheld battery-operated device for detection of CL and common fungal and mycobacterial causes of ulcers.We validated Palm PCR™ for detection of common ulcerative skin pathogens using ATCCPalm PCR™ detected 100% of ATCCPalm PCR™ performs comparably to conventional PCR for detection of Leishmania, fungi, and mycobacteria. This work has implications for the diagnostic approach to tropical ulcers, and has the potential to improve field detection of ulcerative pathogens in resource constrained areas.

The prognosis and treatment of New World tegumentary leishmaniasis is dependent on the infecting species, yet such species identification in the Leishmania Viannia subgenus poses a diagnostic challenge. Currently, speciation relies on standard molecular techniques such as restriction fragment length polymorphism (RFLP) analysis, and Sanger sequencing (SS). Whole-genome sequencing (WGS) is a robust and increasingly cost-efficient tool that may improve Leishmania species identification. We evaluated WGS versus standard RFLP-SS for species identification in three reference and five clinical strains of Leishmania Viannia spp. Internal transcribed spacer1 (its1), cysteine proteinase b (cpb), and heat shock protein 70 (hsp70) polymerase chain reaction-restriction fragment length polymorphism (RFLP) was performed, followed by SS of the its2, cpb, hsp70, and mannose phosphate isomerase (mpi) loci. After de novo assembly, sequences were mapped, and homology compared with both reference strains and reference genomes on National Center for Biotechnology Information. All American Type Culture Collection strains were confirmed to be single-species of L. V. braziliensis, L. V. guyanensis, or L. V. panamensis by WGS. Conversely, RFLP-SS was able to definitively identify one of three isolates to the species level. Clinical samples were identified as either single-species (N = 3), mixed (N = 1), or hybrid (N = 1) infections by WGS, while standard molecular diagnosis required multi-target composite analysis for identification due to loci-dependent results by RFLP-SS. We have corroborated the utility of WGS as a diagnostic tool to speciate members of the L. Viannia subgenus and to discriminate between mixed and hybrid infections. WGS is a potentially useful complement to multistaged RFLP-SS for species identification in Leishmania infections.

RNA virus 1-1 (LRV-1-1) is a dsRNA virus identified in isolates of Leishmania (Viannia) braziliensis and thought to advance localized cutaneous leishmaniasis (LCL) to mucocutaneous or mucosal leishmaniasis (MCL/ML). We examined the prevalence of LRV-1 and its correlation to phenotypes of American tegumentary leishmaniasis caused by L. (V.) braziliensis from Peru to better understand its epidemiology. Clinical isolates of L. (V.) braziliensis were screened for LRV-1 by real-time polymerase chain reaction (PCR) and stratified according to the phenotype: LCL (< 4 ulcers in number) MCL/ML; inflammatory ulcers (erythematous, purulent, painful ulcers with or without lymphatic involvement) or multifocal ulcers (≥ 4 in ≥ 2 anatomic sites). Proportionate LRV-1 positivity was compared across phenotypes. Of 78 L. (V.) braziliensis isolates, 26 (54.2%) had an inflammatory phenotype, 22 (28%) had the MCL/ML phenotype, whereas 30 (38.5%) had LCL. Mucocutaneous or mucosal leishmaniasis was found exclusively in adult male enrollees. Leishmania RNA virus 1 positivity by phenotype was as follows: 9/22 (41%) with MCL/ML; 5/26 (19%) with an inflammatory/multifocal cutaneous leishmaniasis phenotype; and 7/30 (23%) with LCL (P = 0.19). Leishmania RNA virus 1 positivity was not associated with age (P = 0.55) or gender (P = 0.49). Relative LRV-1 copy number was greater in those with MCL/ML than those with inflammatory/multifocal CL (P = 0.02). A direct association between LRV-1 status and clinical phenotype was not demonstrated; however, relative LRV-1 copy number was highest in those with MCL/ML. Future analyses to understand the relationship between viral burden and pathogenesis are required to determine if LRV-1 is truly a contributor to the MCL/ML phenotype.

Background Current drug regimens for cutaneous leishmaniasis (CL) include toxic systemic therapies such as amphotericin B (AB) and pentavalent antimonials. Fluconazole (FZ) is a well-tolerated potential oral alternative for the management CL. To date, few objective data exist to guide clinical decision-making when selecting a therapeutic agent a priori, and standardized, clinically-approved drug susceptibility testing platforms for Leishmania spp. have yet to be established. The Sensititre™ YeastOne™ YO9 plate is a commercialized drug susceptibility plate including AB and FZ used for routine testing of non-fastidious yeast. Our objective was to adapt the readily available Sensititre™ YeastOne™ YO9 plate, to determine drug susceptibility profiles of AB and FZ in cultured isolates of Old World and New World Leishmania spp. for the treatment of CL. Methods Promastigotes were cultured in Tobie’s medium with Locke’s overlay until log phase growth was achieved, inoculated into the Sensititre™ system, and incubated over 96 H. minimum inhibitory concentrations (MICs) were determined colorimetrically, and promastigote death was assessed by conventional microscopy out to 96- h. Colour change correlated to MIC values. Results All strains tested exhibited MIC values for FZ that were ≥ 256 μg/mL. New World strains demonstrated reduced susceptibility to AB (0.25 μg/mL – 0.50 μg/mL AB) compared to Old World strains at 0.12 μg/mL AB (p = 0.02). Seventeen (61%) of 28 Viannia isolates versus 82% (27/33) of non-Viannia isolates were resistant at 0.12 μg/mL AB (p = 0.09). For L. V. braziliensis isolates, mean MIC for AB was 0.375 ± 0.14 μg/mL (range 0.25–0.50 μg/mL), while for isolates of L. V. panamensis it was 0.314 ± 0.26 μg/mL (range 0.12–1.0 μg/mL). Conclusions We adapted the Sensititre™ YeastOne™ YO9 plate for testing of Leishmania spp. susceptibility profiles for commonly used antifungals in the treatment of CL, including AB and FZ. Given its current utility in mycology, optimization of the system for potential clinical implementation in parasitology should be pursued. However evaluation of clinically relevant amastigote-stage stages, and higher concentrations of FZ beyond the upper limit concentration of the Sensititre™ YeastOne™ Y09 plate would be required.

The year 2018 heralded many new developments in the field of tropical medicine, including licensure of novel drugs for novel indications, licensure of existing drugs for existing indications but in novel settings, and globalized outbreaks of both vector-borne and zoonotic diseases. We herein describe five top stories in tropical medicine that occurred during 2018, and illuminate the practice-changing development within each story.

Background Leishmania RNA virus-1 (LRV1) is a double-stranded RNA virus identified in 20–25% of Viannia—species endemic to Latin America, and is believed to accelerate cutaneous to mucosal leishmaniasis over time. Our objective was to quantify known virulence factor (VF) RNA transcript expression according to LRV1 status, causative species, and isolate source. Methods Eight cultured isolates of Leishmania were used, four of which were LRV1-positive (Leishmania Viannia braziliensis [n = 1], L. (V.) guyanensis [n = 1], L. (V.) panamensis [n = 2]), and four were LRV1-negative (L. (V.) panamensis [n = 3], L. (V.) braziliensis [n = 1]). Promastigotes were inoculated into macrophage cultures, and harvested at 24 and 48 h. RNA transcript expression of hsp23, hsp70, hsp90, hsp100, mpi, cpb, and gp63 were quantified by qPCR. Results RNA transcript expression of hsp100 (p = 0.012), cpb (p = 0.016), and mpi (p = 0.022) showed significant increases from baseline pure culture expression to 24- and 48-h post-macrophage infection, whereas hsp70 (p = 0.004) was significantly decreased. A trend toward increased transcript expression of hsp100 at baseline in isolates of L. (V.) panamensis was noted. Pooled VF RNA transcript expression by L. (V.) panamensis isolates was lower than that of L. (V.) braziliensis and L. (V.) guyananesis at 24 h (p = 0.03). VF RNA transcript expression did not differ by LRV1 status, or source of cultured isolate at baseline, 24, or 48 h; however, a trend toward increased VF RNA transcript expression of 2.71- and 1.93-fold change of mpi (p = 0.11) and hsp90 (p = 0.11), respectively, in LRV1 negative isolates was noted. Similarly, a trend toward lower levels of overall VF RNA transcript expression in clinical isolates (1.15-fold change) compared to ATCC® strains at 24 h was noted (p = 0.07). Conclusions Our findings suggest that known VF RNA transcript expression may be affected by the process of macrophage infection. We were unable to demonstrate definitively that LRV-1 presence affected VF RNA transcript expression in the species and isolates studied. L. (V.) guyanensis and L. (V.) braziliensis demonstrated higher pooled VF RNA transcript expression than L. (V.) panamensis; however, further analyses of protein expression to corroborate this finding are warranted. Electronic supplementary material The online version of this article (10.1186/s41182-019-0153-x) contains supplementary material, which is available to authorized users.

Supplementary Methods and Results. (DOCX 24 kb)

Figure S1. Log transformed virulence factor RNA transcript expression in baseline cultures analyzed by grouping strains according to LRV-1 status compared by Mann-Whitney for the following targets: pooled expression (A), cpb (B), gp63 (C), mpi (D), hsp23 (E), hsp70 (F), hsp90 (G) and hsp100 (H). Figure S2. Log transformed virulence factor RNA transcript expression in supernatants post-macrophage infectivity at 24 and 48 h compared by LRV-1 status using t-test for the following targets: cpb-24 h (A), mpi-24 h (B), hsp70–24 h (C), pooled-48 h (D), cpb-48 h (E), and mpi-48 h (F). Figure S3. Log transformed virulence factor RNA transcript expression in baseline cultures analyzed by grouping strains according to species (L. V. panamensis versus other) compared by Mann-Whitney for the following targets: pooled expression (A), cpb (B), gp63 (C), mpi (D), hsp23 (E), hsp70 (F), hsp90 (G) and hsp100 (H). Figure S4. Log transformed virulence factor RNA transcript expression in supernatants post-macrophage infectivity at 24 and 48 h compared by species using t-test for the following targets: pooled VF-24 h (A), cpb-24 h (B), pooled VF-48 h (C), and cpb-48 h (D). Figure S5. Log transformed virulence factor RNA transcript expression in baseline cultures analyzed by grouping strains according to source of cultured isolate (ATCC® versus clinical) compared by Mann-Whitney for the following targets: pooled expression (A), cpb (B), gp63 (C), mpi (D), hsp23 (E), hsp70 (F), hsp90 (G) and hsp100 (H). Figure S6. Log transformed virulence factor RNA transcript expression in supernatants post-macrophage infectivity at 24 and 48 h compared by source of cultured isolate using t-test for the following targets: pooled VF-24 h (A), pooled VF-48 h (B), cpb-48 h (C), and mpi-48 h (D). (DOCX 1229 kb)

Over a 2-year period, common and emerging viruses were documented in >20% of these travelers., Malaria is the most common specific cause of fever in returning travelers, but many other vectorborne infections and viral infections are emerging and increasingly encountered by travelers. We documented common and emerging viral pathogens in malaria-negative specimens from ill travelers returning to Canada. Anonymized, malaria-negative specimens were examined for various viral pathogens by real-time PCR. Samples were positive for herpes simplex viruses 1 or 2 (n = 21, 1.6%), cytomegalovirus (n = 4, 0.3%), Epstein-Barr virus (n = 194, 14.9%), dengue virus types 1–4 (n = 27, 2.1%), chikungunya virus (n = 5, 0.4%), and hepatitis A virus (n = 12, 0.9%). Travel-acquired viral pathogens were documented in >20% of malaria-negative specimens, of which 2.5% were infected with dengue and chikungunya viruses. Our findings support the anecdotal impression that these vectorborne pathogens are emerging among persons who travel from Canada to other countries.

Background: Due to the highly toxic nature of standard drugs used in the treatment of cutaneous leishmaniasis (CL), and overlapping clinical features of CL with ulcers due to fungal and mycobacterial infections, confirmatory diagnostic testing must be undertaken. We evaluated the performance characteristics of a handheld battery operated device for differentiation of Leishmania from known fungal and mycobacterial causes of cutaneous ulcers. Methods & Materials: Using ATCC strains of Leishmania (L. V. braziliensis, L. V. panamensis, L. V. guyanensis), mycobacteria (M. abscessus complex) and fungi (Paracoccidioides brasiliensis), we validated PalmPCR for detection of Leishmania, fungal, and mycobacterial species known to cause cutaneous ulcers.We further validated the device for detection of Leishmania from clinical specimens including filter paper lesion impressions, cytology brushes, and tissue. Respective primers targeted a conserved region of kinetoplast DNA (kDNA) for detection of Leishmania, pan-mycobacterial Hsp65, and pan-fungal ITS3/4 regions. PCR products were visualized using the EGel Go reader, a portable battery-operated system for agarose electrophoresis. Outcome measures were sensitivity and specificity, where conventional end-point or real time PCR was the reference standard. Results: Compared to the reference standard, the PalmPCR device detected 100% of ATCC strains of Leishmania, fungi, and mycobacteria. There was no cross-reactivity of primers with any negative control. The PalmPCR device accurately categorized reference specimens as positive or negative for Leishmania 91.3% of the time (42/46 specimens), yielding sensitivity and specificity of 90% and 91.7%, respectively. Sensitivity of point-of-care PalmPCR for detection of Leishmania in a clinical field setting enrolling patients with suspected CL was 100% compared to reference real time PCR. In 56% of field patients with CL, pan-mycobacterial and/or pan-fungal primers detected co-colonization with species such as Malassezia spp., Aspergillus spp., and Cladosporium spp. In one patient with CL, Mycobacterium doricum was also detected in the ulcer.

Background: Virulence factors (VF) are endogenous molecules that often enhance the pathogenicity of an organism. The role of host age on the immunopathogenesis of leishmaniasis is poorly understood. Despite pediatric populations being frequent victims of leishmaniasis, few studies elucidate whether or not age-specific disease progression patterns exist, on a molecular level in the parasite, whereas clinical epidemiological studies have noted some differences in prognosis and presentation in limited cohorts. Our objective was to quantify known VF RNA transcript expression in clinical isolates, and compare this across age groups. Methods & Materials: Total cellular RNA was extracted from cultured promastigotes of Leishmania , cDNA was reverse transcribed, and qPCR assays were performed to determine transcript expression for: zinc-metalloproteinase ( gp63 ), cysteine proteinase B ( cpb ), mannose phosphate isomerase ( mpi ), and heat shock proteins 23, 70, 83 and 100 ( hsp23 , hsp70 , hsp83 and hsp100 ). Sub-categorical analysis was conducted on a per-gene and pooled basis, between two age groups: pediatric (19 years). Results: Four species isolated from 8 patients with cutaneous leishmaniasis are represented in this study: L. infantum (n = 1, 12.5%), L. tropica (n = 2, 25%), L. V. braziliensis (n = 1, 12.5%) and L. V. panamensis (n = 4, 50%). We did not observe differences in VF RNA transcript expression between pediatric and adult populations for the following: pooled VF (p = 0.68), cpb (p = 0.78), gp63 (p = 0.45), mpi (p = 1.00), hsp23 (p = 1.00), hsp70 (p = 0.57), hsp83 (p = 1.00), and hsp100 (p = 1.00). The same analyses were done for the four L. V. panamensis clinical isolates, with no differences in VF RNA transcript expression: pooled VF (p = 0.67), cpb (p = 1.00), gp63 (p = 1.00), mpi (p = 0.67), hsp23 (p = 0.67), hsp70 (p = 0.67), hsp83 (p = 1.00), and hsp100 (p = 1.00). Conclusion: Here we describe initial quantitative evidence that suggests host age may not be a substantial correlate of VF RNA transcript expression in clinical Leishmania isolates. We failed to elucidate VF RNA transcript expression as the biological underpinning of age-based phenotypic associations suggested by clinical and epidemiological studies. The potential existence of novel or more host-derived immunopathogenesis mechanisms, rather than parasite-specific VFs, may be influenced by age.

Background: Cutaneous leishmaniasis (CL) may present with an inflammatory phenotype, characterized by pain, erythema, and purulent exudate, which is typically treated with antibiotics prior to anti-Leishmania treatment. Although the inflammatory phenotype suggests secondary bacterial infection, bacterial contribution to the pathogenesis of inflammatory CL has yet to be elucidated. The objective of this study was to document the spectrum of bacteria present in inflammatory and non-inflammatory ulcers of CL in order to understand the relationship between disease phenotype and bacterial pathogens. Methods & Materials: Filter paper lesion impressions (FPLIs) from 34 patients with CL (18 inflammatory and 16 non-inflammatory CL) were evaluated via real-time PCR (qPCR) and end-point PCR targeting organisms implicated in skin- and soft-tissue infections, including: Staphylococcus aureus, Enterobacter cloacae, Streptococcus pyogenes, Enterococcus spp. , Citrobacter freundii, Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumonia , as well as 16S rDNA. Positive specimens were Sanger sequenced for species confirmation. Results: Of 34 specimens, 26 (76%) (14 inflammatory and 12 non-inflammatory) had at least one bacterial species detected, and this did not differ by phenotype. Represented species detected include: Staphylococcus aureus (n = 14, 41%), Pseudomonas aeruginosa (n = 9, 26%), Enterobacter cloacae (n = 8, 24%) , Citrobacter freundii (n = 8, 24%) , Enterococcus spp. (n = 6, 18%) , Streptococcus pyogenes (n = 4, 12%) , Klebsiella pneumonia (n = 1, 3%) and Proteus mirabilis (n = 1, 3%). No ulcers were found to harbour Escherichia coli. Prevalence of individual bacterial species did not differ by CL phenotype (p > 0.15). In particular, S. aureus was detected in 6 (37.5%) non-inflammatory ulcers and 8 (44.4%) inflammatory ulcers (p = 1.00). Conclusion: Our findings do not support that bacterial prevalence or individual species composition differs by phenotype of CL ulcers. Further prospective analysis, including WGS of CL ulcers, is warranted to determine the role of empiric antibiotic therapy for those with an inflammatory phenotype of CL.

The ability of vancomycin resistance determinants to be horizontally transferred within enterococci species is a concern. Identification and characterization of vancomycin-resistant enterococci (VRE) in a clinical isolate have a significant impact on infection control practices. In this study, we describe a clinical isolate of Enterococcus gallinarum exhibiting high-level resistance to vancomycin and teicoplanin. The genetic characterization of this isolate showed the presence of vanA and vanB genes in addition to the naturally carried vanC gene. vanA was identified on pA6981, a 35,608-bp circular plasmid with significant homology to plasmid pS177. The vanB operon was integrated into the bacterial chromosome and showed a high level of homology to previously reported Tn 1549 and Tn 5382 . To the best of our knowledge, this is the first report of E. gallinarum carrying both vanA and vanB operons, indicating the importance of identifying the vancomycin resistance mechanism in non- E. faecium and non- E. faecalis enterococcal species.

Backgound. Species of the Leishmania Viannia (L. V.) subgenus harbor the double-stranded Leishmania RNA virus 1 (LRV-1), previously identified in isolates from Brazil and Peru. Higher levels of LRV-1 in metastasizing strains of L. V. guyanensis have been documented in both human and murine models, and correlated to disease severity. Methods. Expression of proinflammatory biomarkers, including interleukin (IL) 1 beta, tumor necrosis factor alpha (TNF-alpha), CXCL10, CCL5, IL-6, and superoxide dismutase, in human macrophages infected with 3 ATCC and 5 clinical isolates of L. V. braziliensis, L. V. guyanensis, and L. V. panamensis for 24 and 48 hours were measured by commercial enzyme immunoassay. Analyses were performed at 24 and 48 hours, stratified by LRV-1 status and species. Results. LRV-1-positive L. V. braziliensis demonstrated significantly lower expression levels of TNF-alpha (P =.01), IL-1 beta (P =.0015), IL-6 (P =.001), and CXCL10 (P =.0004) compared with LRV-1-negative L. V. braziliensis. No differences were observed in strains of L. V. panamensis by LRV-1 status. Conclusions. Compared to LRV-1-negative L. V. braziliensis, LRV-1-positive strains of L. V. braziliensis produced a predominant Th2-biased immune response, correlated in humans to poorer immunologic control of infection and more severe disease, including mucosal leishmaniasis. Effects of LRV-1 on the pathogenesis of American tegumentary leishmaniasis may be species specific.

Background: American tegumentary leishmaniasis (ATL) comprises a discrete set of clinical presentations of leishmaniasis endemic to Latin America and particularly Peru. Leishmania RNA virus-1 (LRV-1) is a double stranded RNA virus identified in 20-25% of the Leishmania Viannia complex, and is believed to be a predictive biomarker of severe ATL. Our objective was to determine the baseline prevalence of LRV-1 and associations with clinical phenotypes of ATL in patients from Peru. Methods & Materials: Banked clinical isolates of Leishmania Viannia spp. from patients residing in or traveling to Peru between 2012 and 2016 were species identified, by PCR, RFLP analysis, and Sanger sequencing, and screened for LRV-1 by real-time PCR. Patient isolates were stratified according to clinical phenotype: localized cutaneous leishmaniasis (LCL) was defined as “non-severe” ATL, whereas “severe ATL” was defined as mucosal or mucocutaneous leishmaniasis (ML/MCL); erythematous, purulent, or painful ulcers and/or lymphatic involvement (inflammatory ulcers); or multifocal/disseminated ulcers (≥4 in ≥2 anatomic sites). Proportions of LRV-1-positivity were calculated and compared across phenotypes. Results: Of 81 patients enrolled, 45(56%) and 36(44%) of ATL cases had the severe and non-severe phenotypes, respectively. Seventeen (38%) of 45 severe ATL cases and 13(36%) of 36 non-severe ATL cases were positive for LRV-1, respectively (p = 1.00). The severe phenotype was over-represented among older patients (median age 42 years vs. 30.5 years in non-severe ATL, p = 0.01), while LRV-1 was over-represented among younger patients (median age 28 years vs. 39 years in LRV-1 negative, p = 0.02). No difference in disease severity by sex (p = 0.12) or causative species (p = 0.057) was identified. Similarly, no difference in LRV-1 status was observed by sex (p = 1.00) or causative species (p = 0.45). LRV-1 copy number was greater in those with ML/MCL compared to those with all forms of CL (p = 0.04). Conclusion: A direct association between LRV-1 status and clinical phenotype was not demonstrated, however LRV-1 copy number was higher in patients with ML/MCL. Our findings suggest age as a contributing factor to disease severity. Given the likelihood of relapse in patients with severe ATL and associated risk of treatment failure, further elucidation of LRV-1's possible contribution to disease severity in ATL warrants further exploration.

Background: Leishmania RNA Virus-1 (LRV-1) is a double stranded RNA virus identified in strains of the Viannia subgenus in Latin America. Virulence factors (VFs) are gene products that augment pathogenicity of an organism, and are mostly constitutively expressed. Both LRV-1 and VFs are known to modulate the host immune response to Leishmania infection. We aimed to quantify RNA transcripts of known VFs in Leishmania to understand the influence, if any, of LRV-1. Methods & Materials: Five clinical cultures of Leishmania Viannia spp. housed in our biobank were species identified by PCR, RFLP analysis, and Sanger sequencing. Cultures were inoculated into a macrophage model and supernatants were collected at 24- and 48-hrs. Total cellular RNA was extracted at baseline and post-macrophage infection, and cDNA was reverse transcribed. LRV-1 status was confirmed by qPCR and quantified using the 2-ΔΔ CT method. qPCR assays were performed using the 18S gene as a reference for the following RNA transcripts: zinc-metalloproteainse (gp63), mannose phosphate isomerase (mpi), cysteine proteinase B (cpb), and heat shock proteins 23, 70, 83, and 100 (hsp23, hsp70, hsp83 and hsp100, respectively). Results: Cultured isolates were derived from male patients with a median age of 22 years (range 9-80 years) and were species identified as: L. V. braziliensis (n = 1, 20%) and L. V. panamensis(n = 4, 80%). Of 5 cultured isolates, 3 (60%) were positive for LRV-1. RNA transcript expression between LRV-1 positive strains did not differ from LRV-1 negative strains for the following VFs at baseline: pooled VFs (p = 0.20), cpb (p = 0.40), gp63 (p = 0.20), mpi (p = 0.20), hsp23 (p = 0.20), hsp70 (p = 0.20), hsp83 (p = 0.20), and hsp100 (p = 0.20). Similarly, transcript expression did not differ by LRV-1 status post macrophage infection for the following: pooled VF at 24- and 48-hrs, respectively (p = 0.80 and p = 0.40), CPB at 48 hrs (p = 0.20) and MPI at 48 hrs (p > 0.99). Conclusion: VF RNA transcript expression did not differ significantly between LRV-1 positive and negative clinical strains of Leishmania at baseline and post-macrophage infection. Our findings suggest that LRV-1 does not contribute to enhanced virulence in our analyzed VFs, however further exploration of additional VFs, particularly those specific to New World or the Viannia subgenus is warranted.